DNA-SYNTHESIS, MICROTUBULE AND NUCLEAR-DYNAMICS IN PORCINE PARTHENOTES

Parthenogenetically activated mammalian oocytes have been used in the past decade as cytoplasts, in an attempt to support the development of nuclear transplant embryos. The present experiments were undertaken t o study the DNA synthesis and the organisation of microtubules, nuclea r envelope and chromatin during the first cell cycle of electrically a ctivated porcine oocytes (parthenotes) matured in vitro by using immun ocytochemistry and laser scanning confocal microscopy. The results sho wed that pronuclear-like (PN) formation began 4-5 h post-activation (h pa), whilst DNA synthesis as revealed by bromodeoxyuridine incorporati on was initiated 5-6 hpa, with a maximum number of labelled oocytes (7 3%) around 11 hpa, and persisted in some parthenotes until 15-16 hpa. In the metaphase II (MII) oocytes, microtubules were detected only in the metaphase II spindle; no lamin A/C antigen was observed. Electrica l DC pulses resulted in 91% of MII oocytes being activated and confoca l microscopy indicated that microtubules were assembled in the spindle first for the extrusion of a second polar body, and for the second ti me for division from one to two cells. Nuclear envelope, indicated by anti-lamin A/C stain, was formed around the time of PN formation and s urrounded the nuclear chromatin of 1- and 2-cell parthenogenotes. Thes e results demonstrate that the apparent normality in both DNA synthesi s and dynamics of microtubules and nuclear envelope is involved with c hromosomal organisation in the parthenotes. In addition, the use of el ectrically activated IVM oocytes for both nuclear transfer and parthen ogenetic studies in pigs is discussed.